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. 2014 Sep 15;27(9):1575-85.
doi: 10.1021/tx500211x. Epub 2014 Sep 2.

Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo

Huadong Chen  1 , Junsheng Fu, Hao Chen, Yuhui Hu, Dominique N Soroka, Justin R Prigge, Edward E Schmidt, Feng Yan, Michael B Major, Xiaoxin Chen, Shengmin Sang
Affiliations

Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo

Huadong Chen et al. Chem Res Toxicol. .

Abstract

In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2(-/-) mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms.

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Figures

Figure 1
Figure 1
6S and its effects on glutathione metabolism, intracellular ROS, and gene expression in HCT-116 cells. (A) Structures of 6S and its cysteine conjugated metabolite, M2. (B) Effect of 6S on cellular GSH/GSSG ratio. HCT-116 cells were treated with 20 μM 6S for 0, 2, 4, 8, or 24 h, and cellular GSH/GSSG was measured using a commercial kit. Each bar represents the mean ± SD of six experiments. *p < 0.0001. (C) Effect of 6S on intracellular ROS. HCT-116 cells were treated with 20 μM 6S for 0, 2, 4, 8, or 24 h, ROS was determined using carboxy-DCFDA, and the starting ROS level was set as 1. *p < 0.0001. (D) A heatmap of differentially expressed genes due to 6S treatment with the brightest green, black, and brightest red of the color scale used for expression values of −3, 0, and +3, respectively. HCT-116 cells were treated with 20 μM 6S for 24 h, and mRNA was isolated for microarray analysis using Agilent two-channel human 8 × 60k microarrays. The heatmap was generated using Cluster 3.0 with data of 11 upregulated and 36 downregulated genes that were identified by SAM. Known Nrf2 target genes are marked with arrows.
Figure 2
Figure 2
Effects of 6S on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of 6S on expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1 in HCT-116 cells. (B) Effect of 6S on the expression of Keap1, Nrf2, and phosphorylated Nrf2 (p-Nrf2). The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after treatment of HCT-116 cells with 6S (20 μM). β-Actin was used as an internal standard. (C) Time-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM 6S for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM 6S for 6 h. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E) IF staining of Nrf2. HCT-116 cells were treated with 20 μM 6S for 12 or 24 h and then fixed and labeled with anti-Nrf2 and appropriate FITC-conjugated secondary antibodies. Cells were counterstained with DAPI for visualization of the nuclei. Slides were viewed using fluorescent microscopy (DAPI, blue; Nrf2, red).
Figure 3
Figure 3
Effects of kinase inhibitors on 6S-induced Nrf2 translocation, phosphorylation, and HMOX1 expression in HCT-116 cells. (A) Effect of kinase inhibitors on 6S induced Nrf2 translocation and phosphorylation. PD098059 (50 μM, a MEK1 inhibitor), LY294002 (50 μM, a PI3K inhibitor), or SB202190 (50 μM, a p38 inhibitor) was used to pretreat the cells for 30 min before they were exposed to 20 μM 6S. After another 24 h of incubation, cytosolic and nuclear Nrf2 as well as p-Nrf2 was determined using western blotting with the appropriate specific antibodies. Lamin B and β-actin were used as internal controls for nuclear and cytosolic extracts, respectively. *p < 0.05. (B) Effect of kinase inhibitors on 6S-induced HMOX1 expression. Inhibitor was used to pretreat the cells for 30 min before they were exposed to 20 μM 6S. After another 24 h of incubation, whole-cell lysates were prepared and assessed for HMOX1 expression by western blotting. *p < 0.05.
Figure 4
Figure 4
6S induced Nrf2 expression in mouse colon. (A) 6S treatment increases nuclear Nrf2 levels in the colon of wild-type (WT) mice. Nrf2 expression is shown by IF and IHC staining. (B) IHC staining of HMOX1 and MT1 in the colon of WT and Nrf2–/– mice after 6S treatment. Scale bar = 50 μm (C) Effects of 6S on HMOX1 and GCLC expression in the colon of WT and Nrf2–/– mice. Colon lysates from five animals of each genotype were analyzed; graph represents the average; *p < 0.05, **p < 0.01. (D) Effect of 6S on the expression of Gclc, Hmox1, and Mt1 mRNA in the colon of WT and Nrf2–/– mice as determined by real-time PCR. *p < 0.05, **p < 0.01.
Figure 5
Figure 5
Effects of M2 on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of M2 on the expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1. (B) Effect of M2 on the expression of Keap1, Nrf2, and p-Nrf2. The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after the treatment of HCT-116 cells with M2 (20 μM). β-Actin was used as internal standard. (C) Time-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM M2 for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM M2 for 6 h. Cytosolic and nucleic Nrf2 were determined using western blotting with the appropriate specific antibodies. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E, F) Effects of kinase inhibitors on M2-induced Nrf2 translocation and phosphorylation (E) and HMOX1 (F) expression. PD098059 (50 μM, a MEK1 inhibitor), LY294002 (50 μM, a PI3K inhibitor), or SB202190 (50 μM, a p38 inhibitor) was used to pretreat the cells for 30 min before they were exposed to 20 μM M2. After another 24 h of incubation, whole-cell lysates were prepared and assessed for Nrf2, p-Nrf2, and HMOX1 expression by western blotting. *p < 0.05.
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References

    1. Leung A. Y., and Foster S. (1996) Encyclopedia of Common Natural Ingredients Used in Food, Drugs, and Cosmetics, 2nd ed., pp 271–274, Wiley, New York.
    1. Jiang H.; Solyom A. M.; Timmermann B. N.; Gang D. R. (2005) Characterization of gingerol-related compounds in ginger rhizome (Zingiber officinale Rosc.) by high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 19, 2957–2964. - PubMed
    1. Jiang H.; Timmermann B. N.; Gang D. R. (2007) Characterization and identification of diarylheptanoids in ginger (Zingiber officinale Rosc.) using high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 21, 509–518. - PubMed
    1. Jiang H.; Xie Z.; Koo H. J.; McLaughlin S. P.; Timmermann B. N.; Gang D. R. (2006) Metabolic profiling and phylogenetic analysis of medicinal Zingiber species: Tools for authentication of ginger (Zingiber officinale Rosc). Phytochemistry 67, 1673–1685. - PubMed
    1. Yu Y.; Huang T.; Yang B.; Liu X.; Duan G. (2007) Development of gas chromatography-mass spectrometry with microwave distillation and simultaneous solid-phase microextraction for rapid determination of volatile constituents in ginger. J. Pharm. Biomed. Anal. 43, 24–31. - PubMed

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